Project summary/Abstract Infectious diseases contribute significantly to human morbidity and mortality. Many pathogens that cause disease in humans, exhibit a very narrow host range, which poses challenges both for study and for developing effective clinical therapies. We will focus our studies on hepatitis B virus (HBV), an archetypal hepatotropic virus with a narrow host range. HBV infections result in chronic infection and can lead to cirrhosis and hepatocellular carcinoma. HBV has an incompletely defined host tropism limited almost exclusively to humans and chimpanzees. While HBV entry and is supported in hepatocytes across a broad variety of species ranging from humans to rodents, HBV replication does not occur in non-human species. We hypothesize that species- specific differences in cell host dependency factors influence viral tropism. The rationale underlying our hypothesis is based on the literature and our own research and preliminary findings showing that HBV infection of mouse hepatocytes engineered with the human HBV entry receptor remain restricted early in the HBV infection. Possible explanations for failure of HBV replication in this model are that there are restriction factors blocking HBV replication, missing factors required for HBV replication, or a combination of both scenarios. To evaluate these possibilities, fusion of human and mouse hepatocytes to produce heterokaryonic cells fully support HBV replication. This preliminary data demonstrates that some missing factor in the mouse cells but present in the human cells is required for HBV replication and completion of the HBV life cycle. Importantly these experiments also verify that a restrictive factor is not present in the mouse hepatocytes. A major goal of this application is to elucidate the human host dependency factors for HBV required for HBV infection. We anticipate that these factors are critical to cell host permissiveness to HBV and will further development of new targets for HBV treatment. Here, we propose to utilize a novel platform to systematically define such species- specific host dependency factors contributing to infection permissiveness. We have engineered mouse hepatoma cell lines with the HBV entry receptor (SLC10A1 or also known as NTCP). We also have a barcoded cDNA library. We have developed a novel HBV reporter virus which maintains HBV genomic structure and organization and importantly viral fitness while enabling expression and tagging by a GFP or luciferase reporter. We have coupled these techniques with new imaging tools which enable us to quantitatively measure HBV RNA and HBV DNA. Using these techniques in combination will enable us to identify the human cell host dependency factors required for HBV replication; the proposed research is innovative, as our current knowledge of the host dependency factors enabling completion of the HBV life cycle has not been elucidated. Overall this work will shed light on the dependency factors critical for the HBV viral life cycle will greatly add to our understanding of the HBV biology. This work will identify new therapeutic targets for HBV treatment.